Alphaviruses are currently being used as a vector platform to develop vaccines for infectious diseases (e.g. see U.S. Pat. Nos. 5,792,462; 6,156,558; 5,811,407; 5,789,245; 6,015,694; 5,739,026; Pushko et al., Virology 239(2): 389-401 (1997), Frolov et al., J. Virol. 71(1): 248-258 (1997); Smerdou and Liljestrom, J. Virol. 73(2): 1092-1098 (1999). Alphaviruses comprise a genus in the Togaviridae, and members of the genus are found throughout the world, in both vertebrate and invertebrate hosts. Among the most studied alphaviruses for vector platforms are Venezuelan Equine Encephalitis (VEE) Virus, Semiliki Forest Virus (SFV), and Sindbis Virus, the prototype member of the genus. Various constructs have been developed to enhance immunogenicity and effectiveness in vaccine applications. Many of these constructs have also been designed to decrease the likelihood of formation of replication-competent virus through recombination. Johnston et al. (U.S. Pat. Nos. 5,792,462 and 6,156,558, cited above) recognized the potential for recombination from a single helper system (in which the complete set of structural proteins of an alphavirus are on one RNA molecule and the nonstructural proteins and gene of interest are on another molecule), and thus designed “double-helper” systems that utilized two helper RNAs to encode the structural proteins. Dubensky et al. (U.S. Pat. No. 5,789,245) and Polo et al. (U.S. Pat. No. 6,242,259) describe the use of two DNA alphavirus structural protein expression cassettes to package alphavirus replicons or other alphavirus vectors. Liljestrom and colleagues have presented data confirming that a “single helper system” will generate wild-type virus particles through recombination (Bergland, et al. 1993 Biotechnology 11(8): 916-920).
By distributing the viral genes among three nucleic acids, two of which comprise the helper system, as in the above-described art, the theoretical frequency of recombination that would create a replication-competent virus is reduced significantly relative to single helper systems. These existing systems include the use of the alphavirus RNA polymerase recognition signals, so that the helper systems can take advantage of the presence of the alphavirus replication machinery for amplification and efficient expression of helper functions. However, the presence of the terminal recognition signals on the helper RNAs also means that recombinants in which the helper constructs are incorporated into the termini of the replicon RNA by RNA recombination remain replicable. It is also recognized (e.g. Liljestrom et al. U.S. Pat. No. 6,190,666, Column 17, lines 45-48) that the capsid binding region of nsP1 is required for the packaging of alphaviral RNA into virus or viral-like particles, and so removal of this region would result in the reduction of packaging (see also Levis et al. 1986 Cell 44:137 and Weiss et al. 1989 J. Virol. 63:530).
Thus, in existing replicon systems, known packaging signals are typically included in replicon RNAs and excluded from helper constructs. However, helper RNAs are nonetheless packaged or copackaged at a lower frequency (Lu and Silver (J. Virol Methods 2001, 91(1): 59-65), and helper constructs with terminal recognition signals will be amplified and expressed in the presence of a replicon, and potentially yield additional recombination events.
The current preferred dosages for administration of vector replicon particles, as described by Johnston et al., or recombinant alphavirus particles, as described by Dubensky et al., are approximately 106 to 108 particles. In the case of chimpanzee administrations, Dubensky et al. have estimated the need for 4 injections, each containing 107-108 particles, with a Sindbis-HBV vaccine. Such dosages require large scale manufacturing procedures, and the amounts produced at such scale may be greater than the predicted frequency for the generation of replication-competent viruses in these existing systems.
Thus, there remains a need to further improve systems for manufacturing alphavirus replicon particles to further reduce the predicted frequency for formation of replication-competent virus, and to optimize manufacturing strategies and costs.